The present invention relates to an alternately targeted adenovirus and includes methods for producing and purifying such viruses as well as protein modifications mediating alternate targeting.
The various physiological responses of a host animal to the presence of a virus depend on the different ways such viruses interact with the host animal, each of which is first mediated by the surface of the virus (xe2x80x9cthe virionxe2x80x9d). The adenoviral virion is a non-enveloped icosahedron about 65-80 nm in diameter (Horne et al., J. Mol. Biol., 1, 84-86 (1959)). It comprises 252 capsomeresxe2x80x94240 hexons and 12 pentons (Ginsberg et al., Virology, 28, 782-83 (1966))xe2x80x94derived from three viral proteins (proteins II, III, and IV) (Maizel et al., Virology, 36, 115-25 (1968); Weber et al., Virology, 76, 709-24 (1977)). Proteins IX, VI, and IIIa, also present, stabilize the virion (Stewart et al., Cell, 67, 145-54 (1991); Stewart et al., EMBO J., 12(7), 2589-99 (1993)).
The hexon provides structure and form to the capsid (Pettersson, in The Adenoviruses, pp. 205-270, Ginsberg, ed., (Plenum Press, New York, N.Y., 1984)), and is a homotrimer of the protein II (Roberts et al., Science, 232, 1148-1151 (1986)). The hexon provides the main antigenic determinants of the virus, and it also contributes to the serotype specificity of the virus (Watson et al., J. Gen. Virol., 69, 525-35 (1988); Wolfort et al., J. Virol., 62, 2321-28 (1988); Wolfort et al., J. Virol., 56, 896-903 (1985); Crawford-Miksza et al., J. Virol., 70, 1836-44 (1996)).
The hexon trimer is comprised of a pseudohexagonal base and a triangular top formed of three towers (Roberts et al., supra; Athappilly et al., J. Mol. Biol., 242, 430-455 (1994)). The base pedestal consists of two tightly packed eight-stranded antiparallel beta barrels stabilized by an internal loop. The predominant antigenic and serotype-specific regions of the hexon appear to be in loops 1 and 2 (i.e., LI or l1, and LII or l2, respectively), within which are seven discrete hypervariable regions (HVR1 to HVR7) varying in length and sequence between adenoviral serotypes (Crawford-Miksza et al., supra).
The penton contains a base, which is bound to the capsid, and a fiber, which is non-covalently bound to and projects from, the penton base. The penton base, consisting of protein III, is highly conserved among serotypes of adenovirus, and (except for the enteric adenovirus Ad40 and Ad41) it has five RGD tripeptide motifs (Neumann et al., Gene, 69, 153-57 (1988)). These RGD tripeptides apparently mediate adenoviral binding to xcex1v integrins, a family of a heterodimeric cell-surface receptors that also interact with the extracellular matrix and play important roles in cell signaling (Hynes, Cell, 69, 11-25 (1992)). These RGD tripeptides also play a role in endocytosis of the virion (Wickham et al. (1993), supra; Bai et al., J. Virol., 67, 5198-3205 (1993)).
The adenoviral fiber is a homotrimer of the adenoviral polypeptide IV (Devaux et al., J. Molec. Biol., 215, 567-88 (1990)), which has three discrete domains. The amino-terminal xe2x80x9ctailxe2x80x9d domain attaches non-covalently to the penton base. A relatively long xe2x80x9cshaftxe2x80x9d domain, comprising a variable number of repeating 15 residue xcex2-sheets motifs, extends outwardly from the vertices of the viral particle (Yeh et al., Virus Res., 33, 179-98 (1991)). Lastly, about 200 residues at the carboxy-terminus form the xe2x80x9cknobxe2x80x9d domain. Functionally, the knob mediates both primary viral binding to cellular proteins and fiber trimerization (Henry et al., J. Virol., 68(8), 5239-46 (1994)). Trimerization also appears necessary for the amino terminus of the fiber to properly associate with the penton base (Novelli et al., Virology, 185, 365-76 (1991)). In addition to recognizing cell receptors and binding the penton base, the fiber contributes to serotype integrity and mediates nuclear localization. Moreover, adenoviral fibers from several serotypes are glycosylated (see, e.g., Mullis et al., J. Virol., 64(11), 5317-23 (1990); Hong et al., J. Virol., 70(10), 7071-78 (1996); Chroboczek et al., Adenovirus Fiber, p. 163-200 in xe2x80x9cThe Molecular Repertoire of Adenoviruses I. Virion Structure and Function,xe2x80x9d W. Doerfler and P. Bxc3x6hm, eds. (Springer, N.Y. 1995)).
Fiber proteins from different adenoviral serotypes differ considerably. For example, the number of shaft repeats differs between adenoviral serotypes (Green et al., EMBO J., 2, 1357-65 (1983)). Moreover, the knob regions from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al., Virology, 186, 280-85 (1992)), and Ad2 and Ad3 fiber knobs are only 20% identical (Signas et al., J. Virol., 53, 672-78 (1985)). In contrast, the penton base sequences of Ad5 and Ad2 are 99% identical. Despite these differences in the knob region, a sequence comparison of even the Ad2 and Ad3 fiber genes demonstrates distinct regions of conservation, most of which are also conserved among the other human adenoviral fibers (see, e.g., FIGS. 1 and 2).
One interaction between the adenoviral virion and the host animal is the process of cellular infection, during which the wild-type virion first binds the cell surface by means of a cellular adenoviral receptor (AR) (e.g., the coxsackievirus and adenovirus receptor (CAR), the MHC class I receptor, etc. (Bergelson et al., Science, 275, 1320-23 (1997); Tanako et al., Proc. Nat. Acad. Sci. (USA), 94, 3352-56 (1997)), Hong et al., EMBO J., 16(9), 2294-06 (1997)). After attachment to an AR, the virus binds xcex1v integrins. Following attachment to these cell surface proteins, infection proceeds by receptor-mediated internalization of the virus into endocytotic vesicles (Svensson et al., J. Virol., 51, 687-94 (1984); Chardonnet et al., Virology, 40, 462-77 (1970)). Within the cell, virions are disassembled (Greber et al., Cell, 75, 477-86 (1993)), the endosome disrupted (Fitzgerald et al., Cell, 32, 607-17 (1983)), and the viral particles transported to the nucleus via the nuclear pore complex (Dales et al., Virology, 56, 465-83 (1973)). As most adenoviral serotypes interact with cells through broadly disseminated cell surface proteins, the natural range of host cells infected by adenovirus is broad.
In addition to cellular infection, host animals react defensively to the presence of adenoviral virions through mechanisms that reduce the effective free titer of the virus. For example, host immune systems, upon exposure to a given adenoviral serotype, can efficiently develop neutralizing antibodies, greatly reducing the effective free titer of the virus upon repeat administration (see, e.g., Setoguchi et al., Am. J. Respir. Cell. Mol. Biol., 10, 369-77 (1994); Kass-Eisler et al., Gene Ther., 1, 395-402 (1994); Kass-Eisler et al., Gene Ther., 3, 154-62 (1996)). Interestingly, such antibodies typically are directed against the same determinants of adenoviral serotype specificity, and are primarily directed to the hypervariable hexon regions and, to some extent, fiber and penton base domains (Watson et al., supra; Wolfort et al. (1988), supra; Wolfort et al. (1985), supra; Crawford-Miksza et al., supra). Of course, the presence of adenoviruses agglutinates red blood cells in humans in a serotype-dependent manner (Hierholzer, J. Infect. Diseases, 123(4), 541-50 (1973)). Additionally, adenoviral virions are actively scavenged from the circulation by cells of the reticuloendothelial system (RES) (see, e.g., Worgall et al., Hum Gene Ther., 8, 1675-84 (1997); Wolff et al., J. Virol., 71(1), 624-29 (1997)). In such a response, Kupffer cells, endothelial liver cells, or other RES cells scavenge the virus from the circulation (see generally, Moghini et al., Crit. Rev. Ther. Drug Carrier Sys., 11(1), 31-59 (1994); Van Rooijen et al., J. Leuk. Biol., 62, 702-09 (1997)). For example, virions can become opsonized, possibly though interaction between collectins and glycocylated viral proteins, triggering recognition by such RES cells; alternatively, such cells may recognize charged amino acid residues on the virion surface (see Hansen et al., Immunobiol., 199(2), 165-89 (1998); Jahrling et al., J. Med. Virol., 12(1), 1-16 (1983)).
Based on the popularity of adenoviruses as gene transfer vectors, efforts have been made to increase the ability of adenovirus to enter certain cells, e.g., those few cells it does not infect, an approach referred to as xe2x80x9ctargetingxe2x80x9d (see, e.g., International Patent Application WO 95/26412 (Curiel et al.), International Patent Application WO 94/10323 (Spooner et al.), U.S. Pat. No. 5,543,328 (McClelland et al), International Patent Application WO 94/24299 (Cotten et al.)). Of course, while the ability to target adenoviruses to certain cell types is an important goal, far more desirable is an adenovirus which infects only a desired cell type, an approach referred to as xe2x80x9calternative targeting.xe2x80x9d However, to exclusively target a virus, its native affinity for host cell ARs must first be abrogated, producing a recombinant adenovirus incapable of productively infecting the full set of natural adenoviral target cells. Efforts aimed at abrogating native adenoviral cell affinity have focused logically on changing the fiber knob. These efforts have proven disappointing, largely because-they fail to preserve the important fiber protein functions of stable trimerization and penton base binding (Spooner et al., supra). Moreover, replacement of the fiber knob with a cell-surface ligand (McClelland et al., supra) produces a virus only suitable for infecting a cell type having that ligand. Such a strategy produces a virus having many of the same targeting problems associated with wild-type adenoviruses (in which fiber trimerization and cellular tropism are mediated by the same protein domain), thus decreasing the flexibility of the vector. Moreover, due to the necessity of having a propagating cell line, and the integral connection between the fiber trimerization and targeting functions, obtaining a mutant virus with substituted targeting is difficult. For example, removing the fiber knob and replacing it with a non-trimerizing ligand (e.g., Spooner et al., McClelland et al., supra) results in a virus lacking appreciable fiber protein.
Aside from the broad natural tropism of the virus noted above, the non-infectious interactions between adenovirus and the host also pose problems for using adenovirus as gene transfer vectors. Such interactions effectively reduce the free titer of a given dose of adenovirus beneath that which is clinically effective. As such, there is currently a need for an adenovirus exhibiting reduced affinity for such natural interactions with a host animal (e.g., target cell affinity, innate or acquired immune survailence, etc). Moreover, there is a need for such a virus which is able to deliver and express a desired transgene within a predefined tissuexe2x80x94an alternatively targeted virus.
The present invention provides a recombinant protein having an amino terminus of an adenoviral fiber protein and having a trimerization domain. A fiber incorporating such a protein exhibits reduced affinity for a native substrate than does a wild-type adenoviral fiber trimer. The present invention further provides an adenovirus incorporating the recombinant protein of the present invention.
The present invention is useful in a variety of gene-transfer applications, in vitro and in vivo, as a vector for delivering a desired gene to a cell with minimal ectopic infection. Specifically, the present invention permits more efficient production and construction of safer vectors for gene transfer applications. The present invention is also useful as a research tool by providing methods and reagents for the study of adenoviral attachment and infection of cells and in a method of assaying receptor-ligand interaction. Similarly, the recombinant fiber protein can be used in receptor-ligand assays and as adhesion proteins in vitro or in vivo. Additionally, the present invention provides reagents and methods permitting biologists to investigate the cell biology of viral growth and infection. Thus, the vectors of the present invention are highly useful in biological research.